Inflammation is a biological response to injury, infection or irritation in which a cascade of cellular and microvascular reactions serves to eradicate the infection, remove damaged tissue and generate new tissue. During inflammation, stromal cells regulate leukocyte recruitment and survival through the secretion of specific chemokines and cytokines. Elimination of the cells and mediators that took part in the inflammatory response is essential for the eventual clearance or resolution of inflammation. Disruption of this homeostasis can lead to chronic inflammation and its corollary of severe illnesses, such as tissue fibrosis. Aberrations in immune responses may also lead to the development of autoimmune diseases, characterized by chronic, detrimental immune reactions in which the host's immune system attacks the host's own tissues.
Fibrosis defines the formation of excess fibrous connective tissue during the reparative and reactive process following tissue damage and inflammation. As major components of fibrosis, pro-fibrotic stromal cells play a critical role in the maintenance of chronic inflammation and often preclude full organ recovery, and in some cases can induce organ failure. This can lead to severe illnesses of major impact on public health such as liver cirrhosis, scleroderma, heart and pulmonary fibrosis, atherosclerosis and asthma, as well as skin fibrosis and scarring. The formation of tissue fibrosis is currently considered to be an irreversible process, which is poorly modulated by anti-inflammatory and immunosuppressive drugs. The mechanisms leading to the formation of tissue fibrosis has remained elusive and therefore has prevented the elaboration of an adequate therapeutic treatment. Nevertheless, it is currently appreciated that macrophages, due to their plastic properties, play indispensable roles at both the initiation of fibrosis and its prevention, through the coordination of the resolution of inflammation and balanced wound healing.
Among the various factors found to take part in inflammatory processes is the 80 kDa iron-binding glycoprotein lactoferrin. This glycoprotein represents one of the first defense systems against pathogens, exhibiting antimicrobial, antibacterial, antiviral and antiparasitic activities. Lactoferrin was also found to influence immune system cells both positively and negatively. On one hand, it has been reported to support proliferation, differentiation and activation of immune cells, and strengthen the immune response. On the other hand, lactoferrin has also been reported to have anti-inflammatory properties, in reducing the production of some pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β) and interleukin 6 (IL-6), among others. In addition, lactoferrin has been reported to mediate inhibition of tumor growth and to possess several other biologic activities, including a ribonuclease activity (capable of RNA hydrolysis) and an osteogenic activity (Kanyshkova et al. Biochemistry 2001, 66(1):1-7).
With the exception of iron binding, the biological activities of lactoferrin are considered to reside in a highly basic domain in the N-terminal region, designated lactoferricin. This part of the protein is released in the stomach at acidic pH by pepsin. Bovine lactoferricin is a highly potent 25aa peptide corresponding to residues 17-41 of lactoferrin, whereas the fragment released from human lactoferrin is larger (including positions 1-41) and has weaker antimicrobial properties. A number of lactoferricin derivatives have been described and tested, which retain at least a part of the activities of the native domain. An antimicrobial peptide derived from ovotransferrin, called OTAP-92, has also been identified, corresponding to positions 109-200 of ovotransferrin (Vogel et al., Biochem Cell Biol. 2002; 80(1):49-63).
Various other lactoferrin fragments were disclosed e.g. by US 2007/0197426, JP 2003/289749, JP 2011006468 and several additional publications to Komine et al. (Mol Immunol. 2007 March; 44(7):1498-508, J Vet Med Sci. 2006 July; 68(7):715-23, J Vet Med Sci. 2006 March; 68(3):205-11 and J Vet Med Sci. 2005 July; 67(7):667-77). These fragments were reported to encompass pro-inflammatory activities. Some of the pro-inflammatory activities of these fragments were suggested to reside within the amino acid sequence phenylalanine-lysine-aspartic acid (FKD), since two of four short synthetic peptides derived from these fragments, namely FKDCHLA and GQKDLLFKDSAI (pep1 and pep4, corresponding to positions 243-249 and 295-307 of human lactoferrin, respectively), demonstrated an inflammation-promoting activity and induction of pro-inflammatory cytokines and chemokines (TNF-α, IL-6, IL-8 and MCP-1). The Komine publications further examined the corresponding bovine synthetic peptides, and reported that bovine pep4 (GQRDLFKDSAL) induced the secretion of TNF-α, IL-6, IL-8 and MCP-1 from bovine mammary gland epithelial lined cells (BMEC), as did its human counterpart; however, bovine pep1 (FKECHLA), differing from human pep1 by replacement of only one amino acid, was found inactive in this system.
Japanese Patent Application Publication No. JP 2004155751 discloses a peptide capable of suppressing the production of inflammatory cytokines such as TNF-α and IL-6, wherein the peptide used may be bovine lactoferrin hydrolyzed with a protease. The publication does not disclose peptides having the sequences FKD or FKE.
WO 2013/014669 to some of the inventors of the present invention discloses lactoferrin fragments of 15, 17 and 23 kDa that are characteristic of inflammation during various stages including resolution. Some of the polypeptide fragments encompass inter alia an FKD and/or FKE sequence. The publication discloses the use of lactoferrin fragments as diagnostic biomarkers for assessing the presence or absence of resolving inflammation and for monitoring the progression of inflammatory resolution in a subject.
Lim et al. (Org. Biomol. Chem., 2008, 6, 1944-1948) refer to an “RGD-FKE” nanoribbon, formed from a cyclic RGD integrin binding motif linked to an oligo-FKFE (Phe-Lys-Phe-Glu) sequence, reported to form a beta strand structure.
Pisarev et al. (Clin Cancer Res 2003; 9:6523-6533) disclose survivin-derived epitopes reactive with immune cells isolated from survivin-immunized subjects, inter alia FKELEGWEP. The publication suggests the use of full length survivin as an anti-cancer vaccine.
U.S. Pat. No. 7,579,436 discloses the use of various peptides, including FKEV, for sulfur and ammonia detection, using a sensory device coated with the peptides. U.S. Pat. No. 7,338,929 discloses immunogenic peptides derived from human IL-13 receptor alpha 2 (IL-13Rα2), inter alia SLDHFKECTV, HFKECTVEY, FKECTVEYEL and DHFKECTV, and a method for immunizing against IL-13Rα2-expressing cancer. U.S. Pat. App. Pub. No. 2012/0021970 discloses the use of the peptide YRFKEHWR for ameliorating mitochondria permeability. Additional polypeptides containing the amino acids FKE are described in WO 2009/027768, EP 0852234, U.S. Pat. No. 8,148,322, U.S. Pat. No. 6,165,730, WO 1999/057144, WO 1995/029938, U.S. Pat. No. 6,303,339, U.S. Pat. No. 5,856,444, U.S. Pat. No. 5,840,513, U.S. Pat. No. 6,664,230 and WO 2012/174117, incorporated herein by reference.
The amino acid sequence of murine lactoferrin comprises the sequence YKE. Certain polypeptides comprising YKE are further described, e.g. by EP 0445801, disclosing the hepatitis C virus epitope RRYKEKEK; U.S. Pat. No. 6,531,130 disclosing ordered peptides comprising a repeated (EYYK)2-6 motif, useful as myelin basic protein agonists for treating or preventing demyelinating autoimmune diseases; WO 95/16462, disclosing EYKEYAEYAEYAEYA as a T cell receptor alpha antigen; U.S. Pat. No. 7,635,480, disclosing KAKPVQKLDDDDDGDDTTYKEERHNK and homologs thereof exhibiting at least 80% similarity useful in preventing, diagnosing and treating leptospirosis; and in WO 03/103572, U.S. Pat. No. 6,660,842 and U.S. Pat. No. 6,607,727, incorporated herein by reference.
None of the art discloses or suggests that an isolated FKE sequence or motif may exhibit an inflammation inhibiting activity. There remains an unmet medical need for providing therapeutic modalities for the treatment of inflammatory and fibrotic diseases.